The food, pharmaceutical and biotechnology industries routinely use a column of agarose gel chromatography (AGC) beads or microparticles having diameters in the range of 50 to 400 .mu.m to characterize, fractionate, and purify important biological products.
In one application, known as gel diffusion chromatography (GDC), gelled agarose microparticles are used to separate proteins or soluble polysaccharides by diffusion according to their molecular size. The porosity of the microparticles is a function of the agarose concentration, which generally ranges from 2 to 10% weight/volume (w/v). The balance of the microparticles (i.e., 90 to 98% w/v) is typically water.
In another application, known as affinity chromatography (AC), the polysaccharide microparticles are derivatized and used to specifically isolate and purify certain biological materials.
A third chromatography application is known as ion exchange chromatography (IEC). IEC is a method of separating a mixture of biopolymers (e.g., proteins, polysaccharides or DNA as a few examples) according to their interaction with an ionic matrix. The ionic matrix can contain anionic moieties (e.g., carboxylate or sulfate groups) and/or cationic moieties (e.g., quaternary ammonium groups). Agarose is rarely used in IEC due to its absence of ionic groups unless derivatized.
Microparticles play an important role in GDC in both fractionating and characterizing the molecular weight of proteins, polysaccharides and DNA. Consequently, the microparticles must meet exacting specifications with regard to purity, spherical geometry, and gel concentration. The gel concentration determines the porosity of the medium and hence its sieving properties. The concentration must be uniform both from microparticle to microparticle in a population and within each gel microparticle. No significant regional dehydration or heterogeneity is tolerable unless it is reversible with rehydration.
The spherical geometry of chromatography media is also important in order to achieve maximum resolution or efficacy in mixture separation. This results from the fact that irregularities in microparticle shape result in a looser packing of the microparticles, which increases the void space (V.sub.o) between microparticles. As can be seen in the following equations, a low V.sub.o is important in achieving the lowest average diffusion coefficient (K.sub.av) for a given substance having an elution volume (V.sub.e). EQU V.sub.t =V.sub.i +V.sub.o +V.sub.p; EQU K.sub.av =(V.sub.e -V.sub.o)/(V.sub.t -V.sub.o),
where V.sub.t is total volume of the microparticle bed, V.sub.i is internal microparticle volume, and V.sub.p is polymer volume. K.sub.av varies from zero, for a substance that is completely excluded from the gel (i.e., V.sub.e =V.sub.o) to almost 1 for a very small molecule which will diffuse throughout the gel, thereby making V.sub.e.apprxeq.V.sub.t.
Thus, as void volume increases for any given volume of packed microparticles, the resolution potential for the bed decreases. One means of increasing the void volume for a given size range of chromatography microparticles is to make the microparticles nonspherical. Such an effect would preclude tight packing as well as adequate porosity within the gel bed thereby causing an increase in void space, elution band broadening and thereby loss of resolution.
The purity of GDC media must be very high since the irreversible inclusion of extraneous organic or inorganic substances in the gelled microparticles will alter both their porosity and affinity characteristics. High gel purity becomes even more important when gel diffusion media are derivatized and converted into AC media. In AC applications, affinities due to extraneous materials within the gel can result in nonspecific binding which compromises the high utility of the AC method. Finally, because GDC and AC media are routinely used as analytical standards for characterizing biomolecules, their uniformity and integrity cannot be compromised.
Conventional methods of forming spherical microparticles for chromatography applications include a microemulsion technique, wherein a heated sol including a thermally-gelling polysaccharide is injected into a heated organic solvent. The sol and the solvent form an emulsion, which is then cooled to allow the sol phase of the emulsion to gel in the form of substantially-spherical microparticles.
Although the microemulsion technique produces microparticles that are generally suitable for chromatography, the technique is not without substantial drawbacks. In particular, the micro-emulsion process for forming gelled microparticles is relatively slow, tedious and costly. Once gelled, the microparticles must then be subjected to an extensive washing process to remove the solvent before the microparticles can be used for chromatography. The inefficiencies generated by these disadvantages increase the cost of the resultant microparticles. Moreover, the organic solvent used to form the microemulsion is a hazardous material which presents health and environmental dangers and requires careful storage, handling and disposal.